Our long-term research goal is to elucidate how human autoimmune diseases are initiated, and are normally prevented. To this end, we have exploited two murine autoimmune ovarian disease (AOD) models. The first is by immunization with the ovarian zona pellucida 3 peptide. The second is to perturb the normal immune system by thymectomy on day 3 of life (D3TX). Our studies in the past 3 years have two major conclusions. First, physiologically- expressed, endogenous antigen (Ag) participates actively in autoimmune disease pathogenesis, with CD4+ CD25- T-cells and autoantibody (autoAb) playing key roles in the process. Second, continuous physiologically-expressed, endogenous Ag also maintains tolerance, and this involves CD4+ CD25+ T-cells. We now propose to define the physiologically-relevant mechanism of tolerance, based on the modulation of autoimmune disease by transgenic Ag-specific CD4+ CD25+ T-cells. We will rely on transgenic mice recently developed in our laboratory: 1) Mice that express transgenic OVA in postpubertal male germ cells, recognized by pOVA (323-339)-specific pathogenic T-cells; and 2) double transgenic mice, expressing testis transgenic OVA, and a pOVA-specific transgenic T-cell receptor (DO11.10). Pre- activated DO11.10 T-cells can induce experimental autoimmune orchitis (EAO) in the OVA-transgenic mice. In addition, the double transgenic mice, which have normal testes, develop EAO following D3TX. These findings strongly indicate that DO11.10 CD25+ T-cells can regulate pathogenic DO11.10 CD25- T-cells in vivo, in the context of autoimmune disease. In Aim 1, we will fully develop the disease models that depend on OVA as autoAg, and the transgenic TCR to pOVA on the responding T-cells. Aim 2 will investigate the hypothesis that autoimmune disease in D3TX mice results from activation of neonatal APC, which promote Ag- specific pathogenic T-cell response, in the presence of endogenous Ag. Aim 3 will test the hypothesis that the Ag- specific regulatory T-cells interact with the effector T-cells in vivo, and that the outcome of the cellular interaction is dependent on endogenous Ag, and clonal anergy of the T-cell populations.